Vaccine

ABSTRACT

The invention provides an attenuated rotavirus population comprising a single variant or substantially a single variant which is defined by a nucleotide sequence encoding at least one of the major viral proteins designated as VP4 and VP7. The invention particularly provides a rotavirus population designated as P43. The invention further provides a novel formulation for a rotavirus vaccine which is in the form of a quick dissolving tablet for immediate dissolution when placed on the tongue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/049,192 filed 6Jun. 2002, which is a 371 application of International ApplicationPCT/EP00/07695, filed 15 Aug. 2000, which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to novel vaccine formulations, methods forpreparing them and their use in therapy. In particular the presentinvention relates to novel rotavirus vaccine formulations.

Acute, infectious diarrhea is a leading cause of disease and death inmany areas of the world. In developing countries, the impact ofdiarrheal disease is staggering. For Asia, Africa and Latin America, ithas been estimated that there are between 3-4 billion cases of diarrheaeach year and of those cases about 5-10 million result in death (Walsh,J. A. et al.: N. Engl. J. Med., 301:967-974 (1979)).

BACKGROUND OF THE INVENTION

Rotaviruses have been recognised as one of the most important causes ofsevere diarrhea in infants and young children (Estes, M. K. Rotavirusesand Their Replication in Fields Virology, Third Edition, edited byFields et al., Raven Publishers, Philadelphia, 1996). It is estimatedthat rotavirus disease is responsible for over one million deathsannually. Rotavirus-induced illness most commonly affects childrenbetween 6 and 24 months of age, and the peak prevalence of the diseasegenerally occurs during the cooler months in temperate climates, andyear-round in tropical areas. Rotaviruses are typically transmitted fromperson to person by the faecal-oral route with an incubation period offrom about 1 to about 3 days. Unlike infection in the 6-month to24-month age group, neonates are generally asymptomatic or have onlymild disease. In contrast to the severe disease normally encountered inyoung children, most adults are protected as a result of previousrotavirus infection so most adult infections are mild or asymptomatic(Offit, P. A. et al. Comp. Ther., 8(8):21-26, 1982).

Rotaviruses are generally spherical, and their name is derived fromtheir distinctive outer and inner or double-shelled capsid structure.Typically, the double-shelled capsid structure of a rotavirus surroundsan inner protein shell or core that contains the genome. The genome of arotavirus is composed of 11 segments of double-stranded RNA which encodeat least 11 distinct viral proteins. Two of these viral proteinsdesignated as VP4 and VP7 are arranged on the exterior of thedouble-shelled capsid structure. The inner capsid of the rotaviruspresents one protein, which is the rotavirus protein designated VP6. Therelative importance of these three particular rotaviral proteins ineliciting the immune response that follows rotavirus infection is notyet clear. Nevertheless, the VP6 protein determines the group andsubgroup antigen, and VP4 and VP7 proteins are the determinants ofserotype specificity.

VP7 protein is a 38,000 MW glycoprotein (34,000 MW whennon-glycosylated) which is the translational product of genomic segment7, 8 or 9, depending on the strain. This protein stimulates formation ofthe major neutralising antibody following rotavirus infection. VP4protein is a non-glycosylated protein of approximately 88,000 MW whichis the translational product of genomic segment 4. This protein alsostimulates neutralising antibody following rotavirus infection.

Since VP4 and VP7 proteins are the viral proteins against whichneutralising antibodies are directed, they are believed to be primecandidates for development of rotavirus vaccines, affording protectionagainst rotavirus illness.

Natural rotavirus infection during early childhood is known to elicitprotective immunity. A live attenuated rotavirus vaccine is thus highlydesirable. Preferably this should be an oral vaccine, as this is thenatural route of infection of the virus.

Early vaccine development for preventing rotavirus infections began inthe 1970s after the discovery of the virus. Initially, attenuatedstrains from animals and humans were studied and had mixed ordisappointing results. More recent efforts have focused on human-animalreassortants that have been more successful.

A rotavirus strain known as 89-12 has been described by Ward; see U.S.Pat. No. 5,474,773 and Bernstein, D. L. et al, Vaccine, 16 (4), 381-387,1998. The 89-12 strain was isolated from a stool specimen collected froma 14 month-old child with natural rotavirus illness in 1988. Accordingto U.S. Pat. No. 5,474,773 the HRV 89-12 human rotavirus was thenculture-adapted by 2 passages in primary African Green Monkey Kidney(AGMK) cells and 4 passages in MA-104 cells as described by Ward in J.Clin. Microbiol., 19, 748-753,1984. It was then plaque purified 3 timesin MA-104 cells (to passage 9) and grown after 2 additional passages inthese cells. One additional passage was made (passage 12) for depositionwith the ATCC under the accession number ATCC VR 2272. The depositedstrain is known as 89-12C2.

The 1998 paper in Vaccine by Bernstein et al is referred to below as theVaccine (1998) paper. The paper describes the safety and immunogenicityof an orally administered live human rotavirus vaccine candidate. Thisvaccine was obtained from strain 89-12, attenuated by passaging withoutplaque purification 26 times in primary AGMK cells and then another 7times in an established AGMK cell line (33 passages in total).

Hereinafter the aforesaid material which has been serially passaged 26times will be referred to as P26 and the material which has beenserially passaged 33 times will be referred to as P33. In general,rotavirus derived by passaging 89-12 n times will be referred to as Pn.

In the examples which follow the P33 material was passaged a further 5times on Vero cells. This is referred to as P38.

The P26 and P33 isolates described in the Vaccine (1998) paper were notdeposited in a culture collection, nor were they analysed to establishtheir genetic characterisation.

It has now been found that the P26 population described in theliterature comprises a mixture of variants. This has been established bygenetic characterisation as described hereinbelow (see examples). P26 istherefore not a reliably consistent population for further passages, inparticular for the production of vaccine lots. Similarly, P33 comprisesa mixture of variants and is not reliably consistent for the productionof vaccine lots.

It has been found that the P26 material is a mixture of at least threeVP4 gene variants. P33 and P38 are similarly a mixture of two variants.These variants appear to be antigenically different, in terms ofneutralising epitopes, to the 89-12C2 strain deposited at the ATCC whenevaluating the neutralizing antibody titers of sera from infantsvaccinated with P33 against these variants. This is illustrated in FIG.3.

Furthermore it has been found that when the P33 material is administeredto infants, two identified variants are replicated and excreted. Of 100vaccinated infants, only 2 showed signs of gastro-enteritis due torotavirus infection, while 20% of a placebo group were infected. Thesefindings suggest that the identified variants are associated withprotection from rotavirus disease. The present invention provides amethod of separating rotavirus variants and an improved live attenuatedrotavirus vaccine derived from a cloned (homogeneous) human rotavirusstrain.

SUMMARY OF THE INVENTION

Accordingly, according to a first aspect the present invention providesan attenuated rotavirus population (isolate), characterised in that itcomprises a single variant or substantially a single variant, saidvariant defined by the nucleotide sequence encoding at least one of themajor viral proteins designated as VP4 and VP7.

Preferably the rotavirus population according to the invention is acloned variant.

By a population comprising a single variant, or substantially a singlevariant, is meant a rotavirus population which does not contain morethan 10%, and preferably less than 5% and most preferably less than 1%of a different variant or variants. Virus populations can be purified tohomogeneity or substantial homogeneity by passaging on suitable celltypes or by performing a series of one or more cloning steps.

An advantage of the invention is that a population comprising a singlevariant is more suitable for the formulation of a consistent vaccinelot. Particular variants defined by nucleotide sequences encoding themajor viral protein may also be associated with enhanced efficacy in theprevention of rotavirus infection.

In one preferred aspect, the single or substantially single variant inthe rotavirus population of the invention is a variant in which the VP4gene comprises a nucleotide sequence comprising at least one of thefollowing: an adenine base (A) at position 788, an adenine base (A) atposition 802 and a thymine base (T) at position 501 from the startcodon.

In a further aspect the single or substantially single variant in thepopulation of the invention is a variant in which the VP7 gene comprisesa nucleotide sequence comprising at least one of the following: athymine (T) at position 605, an adenine (A) at position 897, or aguanine (G) at position 897 from the start codon. Preferably at position897 there is an adenine (A).

In a preferred aspect the single variant in the population according tothe invention has an adenine (A) at positions 788 and 802 and a thymine(T) at position 501 from the start codon in the VP4 gene sequence.

In another preferred aspect the single variant in the populationaccording to the invention has a thymine (T) at position 605 and anadenine/guanine (A/G) at position 897 from the start codon in the VP7sequence. Most preferably in the VP7 sequence there is an adenine (A) atposition 897.

In a particularly preferred aspect the single variant in the populationaccording to the invention has an adenine (A) at positions 788 and 802and a thymine (T) at position 501 from the start codon in the VP4 genesequence, and a thymine (T) at position 605 and an adenine/guanine (A/G)at position 897 from the start codon in the VP7 sequence. Mostpreferably in the VP7 sequence there is an adenine (A) at position 897.

In another aspect the single variant comprises a nucleotide sequenceencoding a VP4 protein wherein the nucleotide sequence is as shown inFIG. 1 (SEQ ID NO: 1), and/or a nucleotide sequence encoding a VP7protein wherein the nucleotide sequence is as shown in FIG. 2 (SEQ IDNO: 2).

The present invention also provides a method of producing a rotaviruspopulation comprising a substantially single variant, the methodcomprising:

-   -   passaging a rotavirus preparation on a suitable cell type;    -   optionally selecting homogeneous culture using the steps of        either:    -   a) limit dilution; or    -   b) individual plaque isolation; and    -   checking for the presence of a substantially single variant by        carrying out a sequence determination of an appropriate region        of the VP4 and/or VP7 gene sequence.

The sequence determination may suitably be carried out by a quantitativeor semi-quantitative hybridisation technique such as slot blothybridisation or plaque hybridisation.

Preferably the selected variant is a variant which is replicated andexcreted when the starting rotavirus preparation is administered to ahuman subject, in particular a child.

The resulting cloned virus population resulting from the methodaccording to the invention may be amplified by further passaging on asuitable cell line.

Suitable cell types for passaging the rotavirus population in the abovemethod include African green monkey kidney (AGMK) cells, which may beestablished cell lines or primary AGMK cells. Suitable AGMK cell linesinclude for example Vero (ATCC CCL-81), DBS-FRhL-2 (ATCC CL-160), BSC-1(ECACC 85011422) and CV-1 (ATCC CCL-70). Also suitable are MA-104(rhesus monkey) and MRC-5 (human - ATCC CCL-171) cell lines. Vero cellsare particularly preferred for amplification purposes. Passaging on Verocells gives a high virus yield.

Techniques for checking whether there is a single variant in a viruspopulation resulting from the method, and for determining the nature ofthat single variant involve standard sequencing or hybridisationprocedures known in the art and are described hereinbelow.

In a preferred aspect the method of the invention is carried out usingan appropriate rotavirus, particularly rotavirus having thecharacteristics of the 89-12 strain or of a passaged derivative thereof.

A particularly preferred single variant population is P43, which wasobtained from P33 (an isolated human rotavirus passages 33 times inculture on appropriate cell types) by a series of end dilution cloningsteps followed by passaging the cloned material on Vero cells foramplification.

A P43 population was deposited at the European Collection of Animal CellCultures (ECACC), Vaccine Research and Production Laboratory, PublicHealth Laboratory Service, Centre for Applied Microbiology and Research,Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom on 13 Aug.1999 under the deposition number 99081301, under the terms of theBudapest Treaty.

Although this indicated public availability is the simplest method ofobtaining the human rotavirus P43, it is not altogether impossible orimprobable that similar and functionally substantially identicalrotaviruses might be produced by these or other methods in view of theteachings of this invention. Such functionally substantially identicalrotaviruses are considered to be biologically equivalent to the humanrotavirus P43 of this invention and therefore are within the generalscope of the present invention. It will therefore be understood that theinvention encompasses rotavirus populations having the characteristicsof the P43 variant as described herein.

It will also be understood that the invention encompasses materialsderived from the deposited P43 ECACC 99081301 by subjecting it tofurther processing such as by propagating it by further passaging,cloning, or other procedures using the live virus or by modifying P43 inany way including by genetic engineering techniques or reassortanttechniques. Such steps and techniques are well known in the art.

Materials derived from the deposited P43 which are covered by theinvention include protein and genetic material. Of particular interestare reassortant rotaviruses which comprise at least one antigen or atleast one segment of P43, for example reassortants which comprise avirulent strain of rotavirus in which one or part of one of the 11genome segments has been replaced by the genome segment or part thereofof P43. Specifically, a rotavirus reassortant in which the segment orpartial segment coding for NSP4 is a P43 segment or partial segment, mayhave useful properties. Reassortant rotaviruses and techniques forpreparing them are well known (Foster, R. H. and Wagstaff, A. J.Tetravalent Rotavirus Vaccine, a review. ADIS drug evaluation, BioDrugs,Gev, 9 (2), 155-178, 1998).

Materials of particular interest are progeny of P43 and immunologicallyactive derivatives of P43. Immunologically active derivatives meansmaterials obtained from or with the P43 virus, particularly antigens ofthe virus, which are capable of eliciting an immune response that isreactive against Rotavirus when injected into a host animal.

In adapting the rotavirus to an appropriate cell line, for example Verocells, it may be necessary to treat the virus so as to get rid of anypotential contaminant such as any adventitious agents that may bepresent and which would otherwise cause contamination. In the case ofether-sensitive adventitious viruses, this may be done by ethertreatment as described hereinbelow. The present invention also relatesto inclusion of such ether treatment as an optional step in the overallprocedure for obtaining an attenuated live rotavirus or vaccineformulated therewith.

Also within the scope of the invention are admixtures of P43 with otherrotavirus variants, for example other cloned variants, or with otherviruses in particular other attenuated viruses. Such mixtures are usefulin the vaccines of the invention which are described hereinbelow.

The present invention also provides a live attenuated rotavirus vaccinewhich comprises a substantially single variant population admixed with asuitable adjuvant or a pharmaceutical carrier.

Preferably, the rotavirus vaccine according to the invention is amonovalent rotavirus vaccine containing a single rotavirus strain.

The present invention is particularly advantageous in providing a liverotavirus vaccine in which the live attenuated rotavirus is a humanrotavirus and does not cause intussusception.

Suitable pharmaceutical carriers for use in the vaccine according to theinvention include those known in the art as being suitable for oraladministration, especially to infants. Such carriers include and are notlimited to carbohydrates, polyalcohols, amino acids, aluminiumhydroxide, magnesium hydroxide, hydroxyapatite, talc, titanium oxide,iron hydroxide, magnesium stearate, carboxymethylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, gelatin,vegetal peptone, xanthane, caraghenane, arabic gum, β-cyclodextrin.

The invention also provides a process for preparing a rotavirus vaccine,for example by freeze drying the virus in the presence of suitablestabilisers or admixing the virus according to the invention with asuitable adjuvant or pharmaceutical carrier.

It may also be advantageous to formulate the virus of the invention inlipid-based vehicles such as virosomes or liposomes, in oil in wateremulsions or with carrier particles. Alternatively or in additionimmunostimulants such as those known in the art for oral vaccines may beincluded in the formulation. Such immunostimulants include bacterialtoxins, particularly cholera toxin (CT) in the form of the holotoxin(entire molecule) or the B chain only (CTB) and the heat labileenterotoxin of E. coli (LT). Mutated LTs (mLTs) which are less likely toconvert to their active form than the native LT are described in WO96/06627, WO 93/13202 and U.S. Pat. No. 5,182,109.

Further immunostimulants which may advantageously be included aresaponin derivatives such as QS21 and monophosphoryl lipid A, inparticular 3-de-O-acylated monophosphoryl lipid A (3D-MPL). Purifiedsaponins as oral adjuvants are described in WO 98/56415. Saponins andmonophosphoryl lipid A may be employed separately or in combination(e.g. WO 94/00153) and may be formulated in adjuvant systems togetherwith other agents. 3D-MPL is a well-known adjuvant manufactured by RibiImmunochem, Montana and its manufacture is described in GB 2122204.

A general discussion of vehicles and adjuvants for oral immunisation canbe found in Vaccine Design, The Subunit and Adjuvant Approach, edited byPowell and Newman, Plenum Press, New York, 1995.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of rotavirus P43 VP4 protein.

FIG. 2 shows the nucleotide sequence of rotavirus P43 VP7 protein.

FIG. 3 shows the neutralizing antibody titers of sera from twelve 4 to 6month old infants vaccinated with P33 against several rotavirus variantsP33, P38, P43 and 89-12C2.

FIG. 4 shows

FIG. 5 shows

FIG. 6 shows

FIG. 7A shows shows a rotavirus vaccine product presentation comprisinga syringe containing the Calcium carbonate antacid buffer (in a liquidform), and a vial containing the lyophilised rotavirus strain.

FIG. 7B shows shows a rotavirus vaccine product presentation comprisinga syringe containing water and a vial containing the lyophilisedrotavirus strain, the calcium carbonate antacid buffer and xanthan.

FIG. 7C shows the lyophilisation, performed directly in a blister, ofrotavirus, CaCO3 and Xanthane gum together.

DETAILED DESCRIPTION OF THE INVENTION

The invention also provides a method for vaccinating human subjects,especially infants, by administering to a subject in need thereof aneffective amount of a vaccine composition according to the invention.Preferably the live attenuated vaccine is administered by oraladministration.

In a preferred aspect the vaccine composition of the invention isformulated with an antacid to minimise inactivation of the vaccine byacid in the stomach. Suitable antacid components include inorganicantacids for example aluminium hydroxide Al(OH)₃ and magnesium hydroxideMg(OH)₂. Commercially available antacids which are suitable for use inthe invention include Mylanta (trade mark) which contains aluminiumhydroxide and magnesium hydroxide. These are insoluble in water and aregiven in suspension.

Aluminium hydroxide is a particularly preferred component of a vaccinecomposition according to the invention as it can provide not only anantacid effect but also an adjuvantation effect.

Also suitable for use as antacids in the vaccine of the invention areorganic antacids such as organic acid carboxylate salts. A preferredantacid in the vaccine composition of the invention contains an organicacid carboxylate salt, preferably a salt of citric acid such as sodiumcitrate or potassium citrate.

A particularly preferred antacid that may be used in the vaccinecomposition of the present invention is the insoluble inorganic salt,calcium carbonate (CaCO₃). The calcium carbonate is able to associatewith the rotavirus and the rotavirus activity is maintained during theassociation with the calcium carbonate.

To prevent sedimentation of calcium carbonate during the filling step,viscous agents are preferably present in the formulation.

Possible viscous agents that may be used include pseudoplasticexcipients. A pseudoplastic solution is defined as a solution havinghigher viscosity on standing compared to its viscosity under agitation.Excipients of this type are natural polymers such as arabic gum,adragante gum, agar-agar, alginates, pectines or semi-synthetic polymersfor example: carboxymethylcellulose (Tyloses C®), methylcellulose(Methocels A®, Viscotrans MC®, Tylose MH® and MB®),hydroxypropylcellulose (Klucels®), and hydroxypropylmethylcellulose(Methocels E® and K®, Viscontrans MPHC®). In general those pseudoplasticexcipients are used together with thixotropic agents. Alternativeviscous agents that may be used are pseudoplastic excipients with lowflowing capacity. Those polymers, at a sufficient concentration, giverise to a structural fluid arrangement resulting in a high viscositysolution having low flowing capacity on standing. A certain quantity ofenergy needs to be given to the system to allow flowing and transfer.External energies (agitation) are needed to destroy temporarily thestructural fluid arrangement in order to obtain a fluid solution.Examples of such polymers are Carbopols® and xanthane gum.

Thixotropic excipents become a gel structure on standing whilst underagitation they form a fluid solution. Examples of thixotropic excipientsare: Veegum® (Magnesium-aluminium silicate) and Avicel RC® (about 89%microcrystalline cellulose and 11% Carboxymethylcellulose Na).

The vaccine composition of the present invention preferably comprises aviscous agent selected from xanthane gum or starch.

Thus the vaccine composition of the present invention is preferablyformulated with a combination of calcium carbonate and xanthane gum.

Other components of a composition used in the invention suitably includesugars for example sucrose and/or lactose.

The vaccine composition according to the invention may containadditional components including for example flavourings (particularlyfor an oral vaccine) and bacteriostatic agents.

Different presentations of the vaccine composition according to theinvention are envisaged.

In one preferred embodiment, the vaccine is administered as a liquidformulation. Preferably the liquid formulation is reconstituted prior toadministration from at least the following two components:

-   -   i) virus component    -   ii) liquid component.

In this embodiment, the virus component and the liquid component arenormally present is separate containers, which may conveniently beseparate compartments of a single vessel, or separate vessels which canbe connected in such a way that the final vaccine composition isreconstituted without exposing it to the air.

Prior to reconstitution, the virus may be in a dry form or a liquidform. Preferably the virus component is lyophilised. Lyophilised virusis more stable than virus in an aqueous solution. The lyophilised virusmay be suitably reconstituted using a liquid antacid composition toproduce a liquid vaccine formulation. Alternatively the lyophilisedvirus may be reconstituted with water or aqueous solution, in which casethe lyophilised virus composition preferably contains an antacidcomponent.

Preferably, the vaccine formulation comprises a virus componentformulated with calcium carbonate and xanthane gum in one compartment orvessel and this is reconstituted with water or aqueous solution presentin the second compartment or vessel.

In another preferred embodiment, the vaccine composition is a solidformulation, preferably a lyophilised cake which is suitable forimmediate dissolution when placed in the mouth. Lyophilised formulationsmay conveniently be provided in the form of tablets in a pharmaceuticalblister pack.

In another aspect the invention provides a rotavirus vaccine in the formof a quick dissolving tablet for oral administration.

In another aspect the invention provides a composition comprising a liveattenuated rotavirus strain, in particular a human rotavirus strain,wherein the composition is a lyophilised solid capable of immediatedissolution when placed in the mouth.

Preferably the quick dissolving tablet according to the inventiondissolves in the mouth of the subject sufficiently quickly to preventswallowing of the undissolved tablet. This approach is particularlyadvantageous for paediatric rotavirus vaccines.

Preferbaly the virus is a live attenuated human rotavirus which isformulated with an inorganic antacid such as calcium carbonate and aviscous agent such as xanthane gum.

A further aspect of the present invention is to provide a lyophilisedformulation wherein the virus component is any rotavirus strain which isformulated with calcium carbonate and xanthane gum.

Vaccines of the invention may be formulated and administered by knowntechniques, using a suitable amount of live virus to provide effectiveprotection against rotavirus infection without significant adverse sideeffects in typical vaccinees. A suitable amount of live virus willnormally be between 10⁴ and 10⁷ ffu per dose. A typical dose of vaccinemay comprise 10⁵-10⁶ ffu per dose and may be given in several doses overa period of time, for example in two doses given with a two-monthinterval. Benefits may however be obtained by having more than 2 doses,for example a 3 or 4 dose regimen, particularly in developing countries.The interval between doses may be more or less than two months long. Anoptimal amount of live virus for a single dose or for a multiple doseregimen, and optimal timing for the doses, can be ascertained bystandard studies involving observation of antibody titres and otherresponses in subjects.

The vaccine of the invention may also comprise other suitable liveviruses for protection against other diseases, for example poliovirus.Alternatively other suitable live virus vaccines for oral administrationmay be given in a separate dose but on the same occasion as therotavirus vaccine composition according to the invention.

Figure Legend for FIG. 3

Sera from twelve 4 to 6 month old infants vaccinated with the P33material as described in the Vaccine (1998) paper were tested forneutralization of P33, P38, P43 and 89-12C2.

The range of neutralization titers of all the tested sera is similar forP33, P38 and P43. The statistical analysis shows no significantdifference in the overall neutralization titers against all threeviruses. This suggests that the conformational and non-conformationalneutralization epitopes of P33, P38 and P43 are equally well recognizedby the anti-P33 sera of P33 vaccinated infants. This observationindirectly suggests that the neutralization epitopes revealed in this invitro assay were not altered between P33, P38 and P43.

The range of neutralization titers of P89-12C2 however significantlydiffers from P33, P38 and P43. This observation suggests that theconformational and non-conformational neutralization epitopes of P33,P38 and P43 are not equally well recognized by the anti-P33 sera of P33vaccinated infants. This observation indirectly suggests that theneutralization epitopes revealed in this in vitro assay were alteredbetween 89-12 C2 and P33, P38 and P43.

The following examples illustrate the invention.

EXAMPLES Example 1 Demonstration That Strain 89-12 at Passage 26 (P26)is a Mixture of Variants

Sequencing of VP4 and VP7 Genes from Different Passage Lots

Sequencing of VP4 and VP7 genes from passage P26 (primary AGMK cells),passage P33 (established (as opposed to primary) AGMK cell line),passage P41 and passage P43 was performed. Total RNA extraction wasreverse transcribed and amplified through PCR in one tube/one step.

Primers Rota 5bis and Rota 29bis amplified the entire VP4 gene andprimers Rota 1 and Rota 2bis amplified the entire VP7 gene. The PCRmaterial has been sequenced using different primers (see Table 1).

The passage P26 sequence differed from the passage P33 sequence by 3bases (at positions 501, 788 and 802 bp from the start codon) in VP4 andby three bases in VP7 (108, 605 and 897 bp from the start codon).

The passage P26 sequence scans of VP4 and VP7 show at mutated positionsthe presence of the passage P33 sequence as a background. Thus it can beseen that passage P26 is a mixture of at least 2 variants.

The passage P33 sequence scans seem homogenous in VP4 and heterogeneousfor VP7 (see Table 2).

Passage P38 (derived from passage 33) was passaged 5 times on Vero cellsand displayed the same set of VP4 and VP7 sequences as passage P33 (AGMKcell line). Thus there was no major change in populations between P33and P38. TABLE 1 Oligonucleotides used for RT-PCR and sequencing NameSequence Sequence ID NO: Position VP7 Rota 1 GGC TTT AAA AGA GAG AAT TTCCGT CTG G (SEQ ID NO:3) −49 to −22 Rota 1bis GGT TAG CTC CTT TTA ATG TATGGT A (SEQ ID NO:4) −16 to 10 Rota 2bis GGT CAC ATC GAA CAA TTC TAA TCTAAG (SEQ ID NO:5) 1014-988 Rota 7 CAA GTA CTC AAA TCA ATG ATG G (SEQ IDNO:6)  266-287 Rota 12 TGT TGA TTT TTC TGT CGA TCC AC (SEQ ID NO:7) 372-394 Rota 46 GGT TGC TGA GAA TGA GAA ATT AGC TAT AGT GG (SEQ IDNO:8)  651-682 Rota 18 CCA CTA TAG CTA ATT TCT CAT TCT CAG CAA CC (SEQID NO:9)  682-651 VP4 Rota 5 TGG CTT CGC CAT TTT ATA GAC A (SEQ IDNO:10)    2-23 Rota 6 ATT TCG GAC CAT TTA TAA CC (SEQ ID NO:11)  878-859Rota 5bis TGG CTT CAC TCA TTT ATA GAC A (SEQ ID NO:12)    2-23 Rota 6bisATT TCA GAC CAT TTA TAA CCT AG (SEQ ID NO:13)  878-856 Rota 25 GGA GTAGTA TAT GAA AGT ACA AAT AAT AG (SEQ ID NO:14)  268-296 Rota 26 CTA TTATTT GTA CTT TCA TAT ACT ACT CC (SEQ ID NO:15)  296-268 Rota 27bis TCGATA CAG TAT AAG AGA GCA CAA G (SEQ ID NO:16)  721-745 Rota 28 TTC ATTAAC TTG TGC TCT CTT ATA CTG (SEQ ID NO:17)  753-727 Rota 31 GTA TAT GTAGAC TAT TGG GAT G (SEQ ID NO:18) 1048-1070 Rota 32 CAT CCC AAT AGT CTACAT ATA C (SEQ ID NO:19) 1070-1048 Rota 45 TGT AAC TCC GGC AAA ATG CAACG (SEQ ID NO:20) 1205-1227 Rota 53 CGT TGC ATT TTG CCG GAG TTA CA (SEQID NO:21) 1227-1205 Rota 54 GTA AGA CAA GAT TTA GAG CGC CA (SEQ IDNO:22) 1465-1487 Rota 55 TGG CGC TCT AAA TCT TGT CTT AC (SEQ ID NO:23)1487-1465 Rota 40 CTT GAT GCT GAT GAA GCA GCA TCT G (SEQ ID NO:24)1703-1727 Rota 39 CAG ATG CTG CTT CAT CAG CAT CAA G (SEQ ID NO:25)1727-1703 Rota 33 CGA TCA TAT CGA ATA TTA AAG GAT G (SEQ ID NO:26)2008-2032 Rota 34 CAT CCT TTA ATA TTC GAT ATG ATC G (SEQ ID NO:27)2032-2008 Rota 29bis AGC GTT CAC ACA ATT TAC ATT GTA G (SEQ ID NO:28)2335-2311

TABLE 2 Oligonucleotides used in hybridization Name Sequence Sequence IDNO. Position VP7 Rota 41 AGT ATT TTA TAC TAT AGT AGA TTA TAT TAA TC (SEQID NO:29) 882-913 Rota 42 AGT ATT TTA TAC TAT GGT AGA TTA TAT TAA TC(SEQ ID NO:30) 882-913 VP4 Rota 15 ATC CCC ATT ATA CTG CAT TCC TTT C(SEQ ID NO:31) 807-783 Rota 16 ATC CCT ATT ATA CTG CAT TTC TTT C (SEQ IDNO:32) 807-783 Rota 35 ATC CCC ATT ATA CTG CAT TTC TTT C (SEQ ID NO:33)807-783 Rota 36 ATC CCT ATT ATA CTG CAT TCC TTT C (SEQ ID NO:34) 807-783

The bases shown in bold type in Table 2 are the sites of specificsequence variation in VP4 and VP7. TABLE 3 sequence variation of VP4 andVP7 genes 3.1 VP4 VP7 501 bp 788 bp 802 bp 108 bp 605 bp 897 bp 167 aa263 aa 268 aa 36 aa 202 aa 299 aa P26 A G/A G/A A C/T A (AGMK) P33 T A AG/A T/C A/G (AGMK) P38 T A A A/G T G/A (VERO) P43 T A A A T A (VERO)

N.B. In a second clone from the 3 clones which were developed to thelevel of production lot, the VP7 897 bp position nucleotide is G, ratherthan A as in the P43 selected clone. This results in a methionine inplace of an isoleucine in the amino acid sequence. Variantscorresponding to both the selected P43 clone and the clone in whichthere is a G in VP7 at 897 bp from the start codon, were excreted in thestools of infants who had been vaccinated with the P33 material.

In Table 3.1, where there are two alternative bases at a particularposition, the first of the two represents the base which appears in amajor population and the second is the base which appears in a minorpopulation. Major and minor variant populations are judged by thestrength of the signal in sequencing. 3.2 VP4 VP7 501 bp 788 bp 802 bp108 bp 605 bp 897 bp 167 aa 263 aa 268 aa 36 aa 202 aa 299 aa P26 LeuGly/Glu Gly/Arg Arg Thr/Met Ile (AGMK) P33 Phe Glu Arg Arg/Arg Met/ThrIle/Met (AGMK) P38 Phe Glu Arg Arg/Arg Met Met/Ile (VERO) P43 Phe GluArg Arg Met Ile (VERO)

Table 3.2 shows the amino acid changes resulting from the nucleotidedifferences between the variants. TABLE 4 VP4 (788-802 positions) VP7(897 position) G-G A-A A-G G-A A G Probes Rota 15 Rota 16 Rota 35 Rota36 Rota 41 Rota 42 Passages P26 − + + + nd nd P33 − + − − ++ + P38 − + −− + ++ P43 − + − − + −Slot Blot Hybridization

The change in populations between passages P26 to P33 on AGMK cells hasbeen further confirmed by slot blot hybridization. The VP4 and the VP7gene fragments generated by RT/PCR were hybridized with oligonucleotideprobes specific for each variant (see Table 3.1 and 3.2). In contrast toP26 which hybridized with Rota 16, Rota 35 and Rota 36 and not with Rota15, the VP4 PCR fragment of the P33 material, at positions 788 and 802hybridized only with Rota 16 and not with either Rota 15 or Rota 35 orRota 36. These results established the presence of at least 3 variantsin P26 (see Table 4).

For the VP7 PCR fragment of the P33 material, position 897 hybridizedwith Rota 41 and Rota 42. These results established the presence of atleast two variants in the P33 material.

Example 2 Isolation and Characterization of the P43 Clone

To isolate P33 components as a homogeneous virus population, threeend-point dilutions of P33/AGMK on Vero cells were performed and theresulting virus was used to infect Vero cells.

Positive wells were selected using two criteria: growth demonstrated bythe largest number of foci detected in the wells and the most isolatedpositive wells on the plates, as is done classically. After 3 enddilution passages in 96 well microtiter plates, 10 positive wells wereamplified successively on Vero cells and evaluated for their yield.

Based on yield, three clones were developed to passage level ofproduction lot. Immunorecognition by polyclonal antibodies was shown tobe similar both between the three clones and between the clones and P33.Homogeneity of the clones was assessed by slot blot hybridization. Thefinal selection of a single clone was based on yield and sequence.

The selected clone was amplified by successive passages on Vero cells togenerate a Master seed, a Working seed and finally production lots.

The selected clone was genetically characterized at different passagelevels by sequencing of VP4 and VP7 (identity) and by specific slot blothybridization of the VP4 and VP7 (homogeneity) of the PCR amplifiedmaterials. The sequence of the VP4 and VP7 genes of the P43 material aregiven in FIGS. 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2) respectively and areidentical to P41.

Homogeneity of the selected clone was assessed by a selectivehybridization using oligonucleotide probes discriminating nucleotidechanges in VP4 and/or VP7 regions for each variant identified duringsequencing of P26/primary AGMK (see Table 4).

The VP4 fragment hybridized with Rota 16 and not with Rota 15, Rota 35or Rota 36.

The VP7 fragment hybridized with Rota 41 and not with Rota 42.

These results confirmed that P43 is a homogeneous population.

Example 3 Removal of Potential Adventitious Virus

Ether was added to P33 (AGMK grown) to a final concentration of 20% for1 hr. Ether was then bubbled out with N₂ for 35 min. No impact on thetitre of P33 seed was observed.

Example 4 Formulation of a Live Attenuated Vaccine

The production lots described above are formulated for oraladministration to infants by the following method.

1. Lyophilised Virus

Standard techniques are used for preparing virus doses. Frozen purifiedviral bulk is thawed and diluted with appropriate medium composition, inthis case Dulbecco's modified eagle Medium, up to a desired standardviral concentration, in this case 10^(6.2) ffu/ml. The diluted virus isthen further diluted with lyophilisation stabiliser (sucrose 4%, dextran8%, sorbitol 6%, amino-acid 4%) up to the target viral titre, in thiscase 10^(5.6) ffu/dose. 0.5 ml aliquots of stabilised virus compositionare aseptically transferred to 3 ml vials. Each vial is then partiallyclosed with a rubber stopper, the sample is freeze dried under a vacuum,the vial is then fully closed and an aluminium cap is crimped in placearound the vial to keep the stopper in place.

For use, the virus is reconstituted using one of the following antacidreconstituents:

(a) Citrate Reconstituent

Sodium citrate is dissolved in water, sterilized by filtration andaseptically transferred into reconstituent containers in 1.5 ml amountsat a concentration of 544 mg Na₃Citrate.2H2O per 1.5 ml dose. Thereconstituent containers may be for example 3 ml vials, or 4 ml vials,or 2 ml syringes, or soft plastic squeezable capsules for oraladministration. As an alternative to maintaining sterile componentsunder sterile conditions, the final container can be autoclaved.

(b) Al(OH)₃ Reconstituent

An aseptic aluminium hydroxide suspension (Mylanta—trademark) isaseptically diluted in sterile water, aseptically transferred toreconstituent containers (for example 2 ml syringes, or soft plasticsqueezable capsules) in 2 ml amounts each containing 48 mg Al(OH)₃. Analternative to using sterile components under sterile conditions is to γirradiate the aluminium hydroxide suspension (preferably at a dilutedstage).

Standard ingredients are included to prevent the suspension fromsettling. Such standard ingredients include for example magnesiumstearate, carboxymethylcellulose, hydroxypropylmethylcellulose,microcrystalline cellulose, and silicone polymers. Bacteriostatic agentsfor example butylparaben, propylparaben or other standard bacteriostaticagents used in food, and flavourings, may also be included.

2. Lyophilised Virus with Al(OH)₃ in Liquid Formulation

Standard techniques are used for preparing virus doses. Frozen purifiedviral bulk is thawed and diluted with appropriate medium composition, inthis case Dulbecco's modified eagle Medium, up to a desired standardviral concentration, in this case 10^(6.2) ffu/ml. Aluminium hydroxidesuspension is added to reach a final quantity of 48 mg/dose and thevirus composition is diluted with lyophilisation stabiliser (sucrose 4%,dextran 8%, sorbitol 6%, amino-acid 4%) up to the target viral titre, inthis case 10^(5.6) ffu/dose. 0.5 ml aliquots of stabilised viruscomposition are aseptically transferred to 3 ml vials. Lyophilisationand closing of the vials is then carried out as described in part 1.

3. Lyophilised Virus with Al(OH)₃ for Blister Presentation

Standard techniques are used for preparing virus doses. Frozen purifiedviral bulk is thawed and diluted with appropriate medium composition, inthis case Dulbecco's modified eagle Medium, up to a desired standardviral concentration, in this case 10^(6.2) ffu/ml. Aluminium hydroxidesuspension is added to reach a final quantity of 48 mg/dose and thevirus composition is diluted with lyophilisation stabiliser which may besucrose, dextran or amino-acid 4%, or gelatin, or vegetal peptone, orxanthane up to the target viral titre of 10^(5.6) ffu/dose. An asepticfilling operation is employed to transfer doses of 0.5 ml or preferablyless to blister cavities. The composition is lyophilised, and theblister cavities are sealed by thermic sealing.

Optionally standard ingredients are included to prevent the aluminiumhydroxide suspension from settling. Such standard ingredients includefor example magnesium stearate, carboxymethylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, and siliconepolymers. Flavourings may also be included.

Example 5 Rotavirus Viral Titration for Various Formulations5.1:Comparison Between Lactose and Sucrose Based Formulations:

Viral titer after Fomulation Viral titer before lyophilisation and 1Batch n° composition lyophilisation week at 37° C. 98G06/01 Lactose: 2%;10^(5.22) 10^(4.67) Dextran: 4%; Sorbitol: 3%; AminoAcids: 2% 98G06/03Sucrose: 2%; 10^(5.28) 10^(4.92) Dextran: 4%; Sorbitol: 3%; AminoAcids:2%

P43 rotavirus was formulated either with sucrose or with lactose asshown in the table above. Viral titration before lyophilisation is theviral titre in the completed formulated liquid (containing sucrosedextran sorbitol aminoacids) and without the lyophilisation step.

Good results are those in which a <0.5 log decrease at thelyophilisation step and <0.5 log decrease during the “1 week at 37° C.”(accelerated stability test) are achieved. The precision of the viraltitration is around + or −0.2 log.

The results indicate that sucrose may be used instead of lactose.

5.2: Effect of Arginine and Replacement of Sorbitol by Maltitol:

Viral titer Viral titer after Fomulation at time = zero lyopjhilisationand 1 Batch n° composition after lyophilisation week at 37° C. 98L16/01Lactose: 2%; 10^(4.8) 10^(4.8) Dextran: 4%; Sorbitol: 3%; AminoAcids: 2%98L16/02 Lactose: 2%; 10^(4.8) 10^(4.9) Dextran: 4%; Sorbitol: 3%;AminoAcids: 2% Arginine: 3% 98L16/04 Lactose: 2%; 10^(4.7) 10⁵   Dextran: 4%; Maltitol: 3%; AminoAcids: 2% Arginine: 3%

The results demonstrate that the addition of arginine (which is known toimprove the stability of the virus during lyophilisation and alsoprovides a basic medium in order to compensate for the stomach acidity)maintains the viral titer.

Sorbitol tends to decrease the glass transition temperature of thelyophilised cake by too great a degree. This can be overcome by usingmaltitol instead of sorbitol as shown above and the viral titer is stillmaintained.

5.3: Various Formulation Compositions

This experiment demonstrates that a number of formulations are possible.Viral titer after Fomulation lyophilisation and 1 Batch n° compositionweek at 37° Viral titer before lyophilisation 99C11/01 Sucrose: 2%;10^(5.24) 10^(5.07) Dextran: 4%; Sorbitol: 3%; AminoAcids: 2% 99C11/02Sucrose: 2%; 10^(5.09) 10^(4.92) Dextran: 4%; Maltitol: 3%; AminoAcids:2% 99C11/04 Dextran: 4%; 10^(4.89) 10^(5.06) Maltitol: 3%; AminoAcids:2% Viral titer at time = zero after lyophilisation 99C17/01 Sucrose: 2%;10^(5.40) 10^(5.41) Dextran: 4%; Sorbitol: 3%; AminoAcids: 2% 99C17/02Sucrose: 2%; 10^(5.30) 10^(4.93) Dextran: 4%; Sorbitol: 1.5%;AminoAcids: 2% 99C17/03 Sucrose: 2%; 10^(5.31) 10^(5.24) Dextran: 4%;AminoAcids: 2% 99C17/04 Sucrose: 2%; 10^(4.42) 10^(4.45) Dextran: 4%;Maltitol: 3%; AminoAcids: 2% 99C17/05 Sucrose: 2%; 10^(4.39) 10^(4.40)Dextran: 4%; Maltitol: 1.5%; AminoAcids: 2% 99C17/06 Sucrose: 2%;10^(5.44) 10^(4.97) Dextran: 4%; Sorbitol: 3%; 99C17/07 Sucrose: 2%;10^(5.11) 10^(4.89) Dextran: 4%; Sorbitol: 1.5%;

5.4: Association Between Rotavirus and Al(OH)₃ Antacid:

Contact time Supernatant Pellets at room viral titer in viral titer inRotavirus Al(OH)₃ H₂O temperature Centrifugation ffu/ml ffu/ml 10^(5.6)ffu/ml 48 mg in 0.76 ml 30 min 8000 rpm, 10^(3.66) 0.240 ml 10 min10^(5.6) ffu/ml 0.48 mg in 0.76 ml 30 min 8000 rpm, 10^(4.41) 0.240 ml10 min 10^(5.6) ffu/ml   1 ml 30 min 8000 rpm, 10^(5.68) 10 minRotavirus in 12 mg in 1.380 ml  30 min 8000 rpm, Below detection10^(4.7) Lyophilised 0.120 ml 10 min Cake

Al(OH)₃ is used as an antacid. This shows that Rotavirus is associatedwith the insoluble inorganic salt (Al(OH)₃) since it centrifugedtogether with the Al(OH)₃ (decrease of viral activity in thesupernatant).

5.5: Dissolution of Al(OH)₃ Antacid by SodiumCitrate Before ViralTitration

Viral samples Dissolution Conditions Viral titers ffu/ml 99B10/06 liquid1.5 ml Na₃Citrate 24 h at room 10^(5.11) formulation before temperaturelyophilisation; 10^(5.43) 99B10/06: 1.5 ml Na₃Citrate 24 h at room10^(4.53) lyophilized 10^(5.43) temperature

When Rotavirus is associated with the Al(OH)₃, it is possible tolyophilise everything (including the Al(OH)₃). After lyophilisation, itis possible to recover the Rotavirus by dissolving Al(OH)₃ inSodiumCitrate. This step does not damage the Rotavirus and retains itsactivity after this dissolution step.

5.6: Infectivity of Rotavirus After Liberation of the Al(OH)₃-RotavirusAssociation:

The mechanism of virus liberation (by dissolution of the carrier) mayvery well occur in vivo. Indeed below pH 6, aluminium hydroxide becomescompletely soluble, and thus, Rotavirus will be liberated in thestomach.Al(OH)₃+3H⁺→Al⁺⁺⁺(water soluble)+3H₂O

In the stomach, Al⁺⁺⁺ ions are not absorbed (J. J. Powell, R.Jugdaohsingh and R. P. H. Thompson, The regulation of mineral adsorptionin the gastrointestinal track, Proceedings of the Nutrition Society(1999), 58, 147-153).

In the intestine, due to the increase of pH, insoluble forms ofaluminium are precipitated (Al(OH)₃ or AlPO₄), and eliminated by thenatural way.

It is unknown whether the newly formed Al(OH)₃ (or AlPO₄) precipitatewill be able to re-associate with free Rotavirus. This raises thequestion of the infectivity of the Al(OH)₃-Rotavirus association itself.

Liberation of Rotavirus from the Al(OH)₃-Rotavirus association by othermechanisms is also possible. Lysine, for example, interferes with theviral adsorption on Al(OH)₃.

Other anions like borate, sulfate, carbonate and phosphate are known tobe specifically adsorbed on aluminium hydroxide, thus, theoretically, itshould be possible to displace (by competition for the adsorption site)Rotavirus from the Al(OH)₃-Rotavirus association.

Thus, Rotavirus may be liberated from the Rotavirus—Al(OH)₃ associationand the liberated Rotavirus remains active.

This liberation can be done either by dissolving Al(OH)₃ (by HCl in thestomach, or by Na₃Citrate in vitro) or by displacing Rotavirus by abasic amino acid (lysine).

5.7: Infectivity of the Al(OH)₃-Rotavirus Association

A single dose of lyophilised Rotavirus was reconstituted with water anddivided into two parts. The first part, considered as the reference,received an additional volume of water. The second part received 24 mgof Al(OH)₃ suspended in 0.240 ml of water (Preclinical viraltitrations).

When Al(OH)₃ is present, Rotavirus is active and the viral titrationvalue is higher compared to the reference sample.

This experiment was repeated without dividing the lyophilised dose, andby adding 12 mg Al(OH)₃ or 24 mg Al(OH)₃.

Here the reference sample was the one reconstituted with aCitrate-Bicarbonate buffer. Thus, the viral titer is again higher in thepresence of Al(OH)₃. DRVC003A46 + DRVC003A46 + 12 mg DRVC003A46 + 24 mg1.5 ml Al(OH)3 Al(OH)3 WL in 0.120 ml + 1.380 ml in 0.240 ml + 1.260 mlbuffer H2O H2O 5.34 6.24 6.05 5.32 5.95 6.26

As in the example above, Rotavirus associates with the Al(OH)₃particles, since the virus can be discarded by centrifugation.DRVC003A46 is a lyophilised formulated Rotavirus (Sucrose:2%; Dextran:4%,Sorbitol:3%; Amino-acids:2%).

SDSAA=Sucrose 2%, Dextran 4%, Sorbitol 3%, Amino-Acid 2%.

According to the viral titration carried out on the supernatant, thequantity of Al(OH)₃ needed to adsorb Rotavirus seems to be low (startingwith one lyophilised dose 5.7 log) scaling Up viral titration): Al(OH)3Adsorption time Titer in supernatant 12 mg 1 hour RT 2.7 24 mg 1 hour RT3.4 48 mg 1 hour RT 3.4 72 mg 1 hour RT 2.0 96 mg 1 hour RT Belowdetection 12 mg Overnight 2.7 24 mg Overnight Below detection 48 mgOvernight 2.5 12 mg Immediate Below detection 24 mg Immediate 2.0 48 mgImmediate Below detection

Time needed to adsorb Rotavirus on Al(OH)₃ seems to be short:

One dose of lyophilised Rotavirus was reconstituted in presence of 24 mgAl(OH)₃, and centrifuged after 0, 15, 60 min and 24 hours. The “culot”were resuspended in SDSAA before viral titration: Time Culot Supernatant0 min 5.26 3.17 15 min 5.34 <1.44 60 min 5.96 <1.44 24 hours 6.13 <1.44

5.8: Using CaCO₃ as Antacid

In order to avoid aluminium in the vaccine, the antacid Al(OH)₃ wasreplaced by another insoluble inorganic salt: CaCO₃ (calcium carbonate).

The phenomena observed with CaCO₃ are parallel to those described forAl(OH)₃:

-   -   Association of Rotavirus with the inorganic salt;    -   Maintainance of Rotavirus activity when associated with the        inorganic salt;    -   Possibility of liberation of Rotavirus from the association by        dissolution of the inorganic base by an acid;    -   Possibility of co-lyophilisation of the antacid and the        Rotavirus.        CaCO₃ and Rotavirus Association

In a first trial, lyophilised Rotavirus (viral titer 5.7) wasreconstituted with a suspension of CaCO₃ in water (50 mg in 1.5 ml); andthen centrifuged, and the viral titer of the supernatant compared to theculot.

This indicates that more that 90% of the Rotavirus is associated withCaCO₃.

Also, when the virus was associated, it was possible to realise thetitration and to recover the original viral quantities.

Also, viral titers are slightly higher that those obtained withoutCaCO₃.

Quantity of CaCO₃ and Rotavirus Association

Lyophilised Rotavirus was reconstituted with a CaCO₃ suspension in water(1.5 ml):

-   -   10 mg    -   50 mg    -   100 mg

and then centrifuged, and the viral titer of the supernatant compared tothe culot. Extempo + Centri. 1 Hour + Centri CaCO3 Culots SurpernatantCulots Surpernatant 100 mg  4.57 3.01 4.79 3.09 50 mg 4.17 4.15 4.223.86 10 mg 3.17 4.77 3.87 4.87

Thus, clearly, more CaCO₃ and more virus is associated, and less isfound in the supernatant. However, the full dose is not completelyrecovered (expected a total of 5.3 at least or even 5.8 as obtainedearlier—see above).

CaCO₃ Protection of Rotavirus During Baby Rossett-Rice Antacid Titration

Using 10 doses of lyophilised Rotavirus (DRVC003A46) and 50mg of CaCO₃,two types of baby Rossett-Rice titration were carried out:

In a classic Rossett-Rice titration, the antacid is mixed with Rotavirusand HCl is poured into this medium.

In the “inverse” baby Rossett-Rice, the situation is the reverse:antacid is dropped into the HCl pool (as it occurs in vivo). Lyophi.Rota Theoretical Measured stored at: Buffer Viral Titer Viral TiterClassical baby Rossett-Rice titration  4° C. 60 mg CaCO3 5.3 4.6 −80° C.60 mg CaCO3 5.3 4.6  4° C. 24 mg Al(OH)3 5.4 <2.9 −80° C. 24 mg Al(OH)35.4 <2.9 Inverse baby Rossett-Rice titration  4° C. 60 mg CaCO3 5.3 4.6−80° C. 60 mg CaCO3 5.3 4.6  4° C. 24 mg Al(OH)3 5.4 <2.9 −80° C. 24 mgAl(OH)3 5.4 <2.9

Thus, in this in vitro experiment, calcium carbonate is able to protectabout 20% of Rotavirus from the presence of HCl, while aluminiumhydroxide is not able to.

5.9: Lyophilisation of Rotavirus in Presence of CaCO₃ Antacid:

Viral titer Viral titer after at time = zero lyopjhilisation and 1 Batchn° Composition after lyophilisation week at 37° C. 99K08/01 Sucrose: 2%10^(5.28) 10^(5.10) Dextran: 4% Sorbitol: 3% Am. Acids: 2% CaCO₃: 50 mg99K08/02 Sucrose: 2% 10^(5.16) 10^(5.15) Dextran: 4% Sorbitol: 3% Am.Acids: 2% CaCO₃: 60 mg 00C24/01 Sucrose: 2% 10^(5.07) 10^(4.69) Dextran:4% Sorbitol: 3% Am. Acids: 2% CaCO₃: 60 mg Xanthane 0.3% 00C24/03Sucrose: 2% 10^(5.07) 10^(4.85) Dextran: 4% Sorbitol: 3% Am. Acids: 2%CaCO₃: 60 mg Xanthane 0.3% 00E09/25 Sucrose: 2% 10^(5.03) 10^(4.91)Dextran: 4% Sorbitol: 3% Am. Acids: 2% CaCO₃: 60 mg Xanthane 0.25%00E09/30 Sucrose: 2% 10^(5.01) 10^(4.87) Dextran: 4% Sorbitol: 3% Am.Acids: 2% CaCO₃: 60 mg Xanthane 0.30% 00F26/06 Sucrose: 2% 10^(4.50)10^(4.70) Dextran: 4% Sorbitol: 3% Am. Acids: 2% CaCO₃: 60 mg Starch: 2%

This is the “all in one”—lyophilisation of Rotavirus and antacid (CaCO₃)together in the same vial. To prevent sedimentation of CaCO₃ during thefilling step, viscous agents are needed. Examples of such viscous agentsinclude Xanthane gum and Starch. The Rotavirus activity is maintainedeven in the presence of Xanthane gum and Starch.

5.10 Lyophilised Tablets for Quick Disintegration When Placed in theMouth:

The following formulations demonstrate the “lyoc” concept. That is,quick dissolution of the lyophilised cake in the mouth. Viral titerafter lyopjhilisation and 1 Batch n^(o) Fomulation composition week at37° Viral titer before lyophilisation 99B10/06 Sucrose 4% 10^(5.11)10^(4.53) Sodium glutamate 3.7% Al(OH)3 48 mg 99C11/12 Maltitol 3%10^(4.16) 10^(3.79) Al(OH) 48 mg Hydroxypropylmethyl- cellulose: 1%Viral titer at time = zero after lyophilisation 00C24/05 Sucrose: 2%10^(5.02) 10^(4.54) Dextran: 4% Sorbitol: 3% Am. Acids: 2% CaCO₃: 60 mgXanthane 0.3% 00C24/06 Sucrose: 2% 10^(4.86) 10^(4.56) Dextran: 4%Sorbitol: 3% Am. Acids: 2% CaCO₃: 60 mg Xanthane 0.3% 00F26/11 Sucrose:1% 10^(4.70) 10^(4.40) Dextran: 2% Sorbitol: 1.5% Am. Acids: 1% CaCO₃:60 mg Starch: 2%

In the “lyoc concept” both Xanthane and Starch can be used (maintainingthe quick dissolution properties of the lyophilised cake).

Example 6 Use of Calcium Carbonate as the Antacid for the RotavirusVaccine Composition

When a suspension of CaCO₃ in water is used as the antacid for Rotavirusthere is a problem that the calcium carbonate particles sediment rapidlywhen placed in water since the powder density value approaches 2.6 andthe average particle size is 30 μm.

This sedimentation can be slowed by:

-   -   1 increasing the density of the surrounding medium    -   2 increasing the viscosity of the surrounding medium    -   3 reducing the particles size    -   4 keeping particles away from each other

6.1: Increasing Density of the Surrounding Medium:

When the CaCO₃-Water suspension (when placed in the syringe) is placedon the lyophilised cake (containing sucrose 2%, dextran 4%; sorbitol 3%;amino-acids 2%) the density of the surrounding medium is increased, butthe speed of CaCO₃ sedimentation is not very much different from theCaCO₃-Water Suspension.

6.2 Increasing the Viscosity of the Surrounding Medium:

Pseudoplastic Excipents

A pseudoplastic solution is defined as a solution having higherviscosity on standing compared to its viscosity under agitation.

Usual excipients of this type are:

-   -   natural polymers for example:        -   arabic gum        -   adragante gum        -   agar-agar        -   alginates        -   pectines    -   semi-synthetic polymers for example:        -   carboxymethylcellulose (Tyloses C®)        -   methylcellulose (Methocels A®, Viscotrans MC®, Tylose MH®            and MB®)        -   hydroxypropylcellulose (Klucels®)        -   hydroxypropylmethylcellulose (Methocels E® and K®,            Viscontrans MPHC®)

In general those pseudoplastic excipients are used together withthixotropic agents.

Pseudoplastic Excipients With Low Flowing Capacity

Those polymers, at a sufficient concentration, give rise to a structuralfluid arrangement resulting in a high viscosity solution having lowflowing capacity on standing. A certain quantity of energy needs to begiven to the system to allow flowing and transfer.

External energies (agitation) are needed to destroy temporarily thestructural fluid arrangement in order to obtain a fluid solution.

Examples of such polymers are Carbopols® and Xanthane gum.

Thixotropic Excipents

With these excipents, on standing, a gel structure is obtained; whileunder agitation a fluid solution is obtained.

Examples of thixotropic excipients are: Veegum ®(Magnesium-aluminiumsilicate) and Avicel RC® (about 89% microcrystalline cellulose and 11%Caboxymethylcellulose Na).

6.3 Reducing the Particles Size

A reduction in the CaCO₃ particle size resulted in a decrease in theantacid capacity of the compound.

6.4 Keeping Particles Away From Each Other

This is the case in Veegum® and Avicel® for which insoluble particlessmaller (about 1 μm) than the CaCO₃ particles, are placed between CaCO₃particles in order to prevent aggregation.

Example 7 Product Design

The following schemes demonstrate examples of possible product designs.

7.1 CaCO₃ in the Syringe

Having already clinical batches of Rotavirus in lyophilised vials, theantacid can be placed in the reconstituent liquid contained in thesyringe.

In this product presentation, sedimentation of CaCO₃ must be undercontrol not only during the filling steps, but also during the completeshelf-live of the product (at least 2 years).

7.2 CaCO₃ in the Lyophilised Vial 7.3. Lyophilisation in a Blister

In this case Rotavirus, CaCO₃ and Xanthane gum are lyophilised togetherdirectly in the blister.

Example 8 Lyophilisation of Different Strain of Rotavirus

Viral titer at t = zero Viral titer after after lyopjhilisation andBatch n^(o) Rotavirus strain Fomulation composition lyophilisation 1week at 37° 00F26/01 G1 Sucrose: 2% 10^(4.6) 10^(4.7) SB purif n^(o)61Dextran: 4% PRO/0232 Sorbitol: 3% Am. Acids: 2% 00F26/02 G2 (DS-1)Sucrose: 2% 10^(4.4) 10^(4.4) Dextran: 4% Sorbitol: 3% Am. Acids: 2%00F26/03 G3(P) Sucrose: 2% 10^(4.6) 10^(4.5) Dextran: 4% Sorbitol: 3%Am. Acids: 2% 00F26/04 G4 (VA-70) Sucrose: 2% 10^(4.8) 10^(4.8) Dextran:4% Sorbitol: 3% Am. Acids: 2% 00F26/05 G9 (W161) Sucrose: 2% 10^(4.6)10^(4.5) Dextran: 4% Sorbitol: 3% Am. Acids: 2%

The strains DS-1, P and VA70 are described as Human rotavirus referencestrains for serotype G2, G3 and G4 respectively at page 1361 of “Fields”Raven press 1990, second edition.

In this experiment different Rotavirus strains have been lyophilised.For all, both the viral titer have been maintained during lyophilisationand accelarated stability (one week at 37° C.) has been shown.

Example 9 Phase I Safety Study in Adults of One Oral Administration ofthe Rotavirus Vaccine.

A Phase I study was carried out to assess the safety and reactogenicityof a single oral dose of 10^(6.0) ffu of the P43 vaccine in healthyadults aged 18 to 45 years.

The clinical trial was double blind and randomized. It wasplacebo-controlled and self-contained. The study was performed in onesingle centre in Belgium.

Study Population

A total of 33 subjects, 11 in the placebo group and 22 in the vaccinegroup, were enrolled and all completed the study. All volunteers wereCaucasians. Their mean age at the time of vaccination was 35.3 years,with a range of 18 to 44 years. The trial began in January and ran forjust over one month.

Material

Vaccine

Clinical lots of P43 vaccine were produced, purified, formulated andlyophilized according to Good Manufacturing Practices. The lots werereleased by Quality Control and Quality Assurance. Each vial of vaccinecontained the following components:

Active Ingredient: P43 strain Min. 10^(5.8) ffu

Excipients, Stabilizers: Sucrose 9 mg Dextran 18 mg  Sorbitol 13.5 mg  Amino acids 9 mgPlacebo

Vials of placebo were prepared and released. Each vial of placebocontained the following components:

Excipients, Stabilizers: Sucrose 9 mg Dextran 18 mg  Sorbitol 13.5 mg  Amino acids 9 mgDiluent

Water for injection was used as diluent to reconstitute vaccine andplacebo.

Administration

Approximately 10 to 15 minutes before administration of the vaccine orthe placebo, subjects of both groups were given 10 ml of Mylanta®orally. Mylanta® is a registered antacid. The antacid increases the pHof the stomach and prevents inactivation of the rotavirus during itspassage through the stomach.

To prepare the vaccine, two vials of lyophilized P43 containing 10^(5.8)ffu per vial were reconstituted with 1.5 ml of diluent water forinjection. This achieved a calculated viral titer of 10^(6.1) ffu perdose. The reconstituted vaccine was administered promptly as a singleoral dose.

To prepare the placebo, two vials of lyophilized placebo werereconstituted with 1.5 ml water for injection and administered orally asa single dose.

Safety and Reactogenicity

The following criteria of safety and reactogenicity applied:

Solicited general symptoms were fever, diarrhea, vomiting, nausea,abdominal pain and loss of appetite. They were recorded during eightdays post administration.

Unsolicited symptoms were recorded during 30 days post administration.

Serious adverse events were recorded during the entire study period.

Diarrhea samples were to be collected during eight days postadministration.

The results were:

No solicited symptoms, no unsolicited and no serious adverse events werereported during the respective observation periods.

No cases of diarrhea were reported.

Conclusions

SB Biologicals P43 vaccine was safe relative to the placebo whenadministered orally in a double-blind fashion as a single dose at thedose of 10^(6.1) ffu to healthy adult volunteers aged 18 to 44.

1. A method of producing a purified human rotavirus populationcomprising a substantially single variant, the method comprising:passaging a rotavirus preparation on a suitable cell line; selecting ahomogeneous culture using at least one of: limit dilution; andindividual plaque isolation; and identifying a substantially singlevariant by sequencing at least a portion of one or both of a VP4 and aVP7 gene sequence.
 2. The method of claim 1, wherein the rotaviruspreparation is derived from a human rotavirus infection.
 3. The methodof claim 1, comprising passaging the rotavirus preparation on cellsselected from: AGMK cells and VERO cells.
 4. The method of claim 2,comprising passaging the rotavirus preparation on VERO cells.
 5. Themethod of claim 1, comprising selecting a homogeneous culture by limitdilution.
 6. The method of claim 1, comprising identifying asubstantially single variant encoding a VP4 protein comprising at leastone of a phenylalanine (Phe) at position 167, a glutamic acid (Glu) atposition 263, and an arginine (Arg) at position
 268. 7. The method ofclaim 6, comprising identifying a substantially single variant encodinga VP4 protein comprising a phenylalanine (Phe) at position 167, aglutamic acid (Glu) at position 263, and an arginine (Arg) at position268.
 8. The method of claim 6, comprising identifying a substantiallysingle variant comprising a nucleotide sequence encoding VP4 comprisingat least one of: an adenine base (A) at position 788; an adenine base(A) at position 802; and a thymine base (T) at position
 501. 9. Themethod of claim 8, comprising identifying a substantially single variantcomprising a nucleotide sequence encoding VP4 comprising an adenine base(A) at position 788; an adenine base (A) at position 802; and a thyminebase (T) at position
 501. 10. The method of claim 1, comprisingidentifying a substantially single variant encoding a VP7 proteincomprising at least one of: a methionine (Met) at position 202, and anisoleucine (Ile) at position
 299. 11. The method of claim 10, comprisingidentifying a substantially single variant encoding a VP7 proteincomprising a methionine (Met) at position 202, and an isoleucine (Ile)at position
 299. 12. The method of claim 10, comprising identifying asubstantially single variant comprising a nucleotide sequence encodingVP7 comprising at least one of: a thymine (T) at position 605; anadenine (A) at position 897; and a guanine (G) at position
 897. 13. Themethod of claim 12, comprising identifying a substantially singlevariant comprising a nucleotide sequence encoding VP7 comprising athymine (T) at position 605; an adenine (A) at position 897; and aguanine (G) at position
 897. 14. The method of claim 1, comprisingselecting a homogenous culture with the characteristics of rotavirusstrain 89-12 or a derivative thereof.
 15. The method of claim 1, whereinthe homogenous culture comprises a single variant that replicates and isexcreted by humans.
 16. The method of claim 1, further comprisingtreating the preparation with ether to remove adventitiousether-sensitive contaminating agents.
 17. A purified human rotaviruspopulation comprising a substantially single variant produced by themethod of claim
 1. 18. An immunogenic composition comprising thepurified human rotavirus population of claim 17 in admixture with anantacid and a viscous agent.